Project Summary Hepatoblastoma (HB) is the most common liver tumor in young children, and hepatocellular carcinoma (HCC), which is associated with very poor prognosis, is more common in older children; interestingly, middle-childhood tumors often histologically resemble HBs but contain genomic features of HCCs. Predictive molecular biomarkers are needed to distinguish between these tumors, HBs and HCCs. We hypothesize that HBs with HCC features (HB-HCC) are intermediary-stage tumors and precursors to the more proliferative HCCs that eventually overwhelm other tumor subclones. Molecular profiling of multiple biopsies of these tumors will reveal their clonal composition, and the alterations that characterize HBs and HCCs and may transform HBs to HCCs. We propose to identify biomarkers that are specific to HBs, HB-HCCs, and HCCs in young children, to determine their clonal composition and to infer whether HB-HCCs?and HCCs?develop from or share common ancestry with HBs. In preliminary work, we identified and validated biomarkers that are specific to low- and high-risk HBs and that can inform therapeutics for children with HB. We also developed technology for reconstructing tumor evolution from genetic profiles of multiple tumor sections. Building on these two efforts we propose to sequence the exomes of four HB-HCCs of consented middle-childhood patients using 6 biopsies per tumor at high-coverage to identify genomic alterations, predict the clonal composition of each biopsy, and reconstruct tumor clonal phylogeny. By comparing the architecture of these tumors to each other and to existing profiles of thirty-four HBs and twenty pediatric HCCs we will identify common and differentiating biomarkers for HB-HCCs. Conceptual innovations in our proposal include exploring the link between HBs and HCCs to better diagnose high-risk HBs through the study of an intermediary tumor type. Technological innovations include the use of new analysis methods to interpret the function of genetic alterations observed in HB- HCCs. In summary, we propose to identify predictive biomarkers that will differentiate lower-risk HBs from high-risk HBs and HCCs to improve risk stratification and, consequently, improve therapies for all children with hepatocellular neoplasms. This is the greatest challenge for improving treatment of hepatocellular neoplasms and has the potential to improve outcome for both low- and high-risk pediatric liver cancer patients.